The present invention relates to ion fragmentation techniques by electron-ion reactions in multipolar radiofrequency fields like those in quadrupole ion traps or in ion guides, and devices to perform ion fragmentation by such techniques. The fragmentation techniques are useful for tandem mass spectrometry.
Mass spectrometry is an analytical technique by which ions of sample molecules are produced and analyzed according to their mass-to-charge (rnz) ratios. The ions are produced by a variety of ionization techniques, including electron impact, fast atom bombardment, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Analysis by m/z is performed in analysers where the ions are either trapped for a period of time or fly through towards the ion detector. In the trapping analysers, such as quadrupole ion trap (Paul trap) and ion cyclotron resonance (ICR cell or Penning trap) analysers, the ions are spatially confined by a combination of magnetic, electrostatic or alternating electromagnetic fields for a period of time typically from about 0.1 to 10 seconds. In the transient-type analysers, such as magnetic, quadrupole filter and time-of-flight analysers, the residence time of ions is shorter, in the range of about 1 to 100 xcexcs.
Tandem mass spectrometry is a general term for mass spectrometric methods where sample ions of desired mass-to-charge are selected and dissociated inside the mass spectrometer and the obtained fragment ions are analyzed according to their mass-to-charge ratios. Dissociation of mass-selected ions can be performed in a special cell between two rnz analysers. The cell is usually based on a multipole, i.e. quadrupole, hexapole, etc. ion guide. In trapping instruments, dissociation occurs inside the trap. Tandem mass spectrometry can provide much more structural information of the sample molecules.
To fragment ions inside the mass spectrometer, collisionally-induced dissociation (CID) is most commonly employed. In the predominant technique, the m/z-selected ions collide with gas atoms or molecules, such as e.g. helium, argon or nitrogen, with subsequent conversion of the collisional energy into internal energy of the ions. Alternatively, ions may be irradiated by infrared photons (infrared multiphoton dissociation, IRMPD), which also leads to the increase of the internal energy. Ions with high internal energy undergo subsequent dissociation into fragments, one or more of which carry electric charge. The mass and the abundance of the fragment ions of a given kind provide information that can be used to characterize the molecular structure of the sample in question.
Both collisional and infrared dissociation techniques have serious drawbacks. Firstly, low-energy channels of fragmentation dominate, which can reduce the multiplicity of bond cleavages and thus the fragmentation-derived information. Even at relatively low energy CID conditions xe2x80x9cweaklyxe2x80x9d bonded functional groups are easily detached and therefore structural information can be limited. The presence of easily detachable groups results in the loss of information on their location. Finally, both collisional and infrared dissociations become ineffective for large molecular masses.
To at least partially overcome these problems, a number of ion-electron dissociation reactions has recently been proposed (see review Zubarev, Mass Spectrom. Rev. 22 (2003) 57-77). One of such reactions is electron capture dissociation (ECD) (Zubarev, Kelleher and McLafferty, J. Am. Chem. Soc. 120 (1998) 3265-3266).
The ECD technique is technically related but physically different from earlier work of using high-energy electrons to induce fragmentation by collisions with electrons (Electron Impact Dissociation, EID). U.S. Pat. No. 4,731,533 describes the use of high-energy electrons (about 600 eV) that are emitted radially on an ion beam to induce fragmentation. Similarly, U.S. Pat. No. 4,988,869 discloses the use of high-energy electron beams 100-500 eV, transverse to a sample ion beam to induce fragmentation. The method suffers from low efficiency, with a maximum fragmentation efficiency for parent ions of about 5%.
In contrast to EID, in the ECD technique positive multiply-charged ions dissociate upon capture of low-energy ( less than 1 eV) electrons in an ion cyclotron resonance cell. The low-energy electrons are produced by a heated filament, or by a dispenser cathode (Zubarev et al., Anal. Chem. 73 (2001) 2998-3005). Electron capture can produce more structurally important cleavages than collisional and infrared dissociations. In polypeptides, for which mass spectrometry analysis is widely used, electron capture cleaves the N-Ca backbone bonds (so called c or z type fragmentation), while collisional and infrared excitation cleaves the amide backbone bonds (peptide bonds, so called b or y type fragmentation). Combination of these two different types of cleavages provides additional sequence information (Horn, Zubarev and McLafferty, Proc. Natl. Acad. Sci. USA, 97 (2000) 10313-10317). Moreover, disulfide bonds inside the peptides that usually remain intact in collisional and infrared excitations, fragment specifically upon electron capture. Finally, some easily detachable groups remain attached to the fragments upon electron capture dissociation, which allows for determination of their positions. This feature is especially important in the analysis of post-translational modifications in proteins and peptides, such as phosphorylation, glycosylation, y-carboxylation, etc.
Other ion-electron fragmentation reactions also provide analytical benefits. Increasing the electron energy to 3-13 eV leads to hot-electron capture dissociation (HECD), in which electron excitation precedes electron capture. The resulting fragments undergo secondary fragmentation, which allows for distinguishing between the isomeric leucine and isoleucine residues (Kjeldsen, Budnik, Haselmann, Jensen, Zubarev, Chem. Phys. Lett. 356 (2002) 201-206). In electron detachment dissociation (EDD) (Budnik, Haselmann and Zubarev, Chem. Phys. Lett. 342 (2001) 299-302), 20 eV electrons ionize peptide di-anions, which produces effect similar to ECD. EDD is advantageous for acidic peptides and peptides with acidic modifications, such as sulfation.
The drawback of current ion-electron fragmentation methods lies primarily in that they are only efficient in Penning ion traps, which are not in widespread use due to their cost and technical complexity. In the much more widespread Paul traps, multipole collisional cells and ion guides, the radiofrequency (rf) electric field with the typical amplitude of 500 V and frequency of 1 MHz rapidly deflects the electrons or increases their energy above the region of 20 eV, below which the ion-electron reactions are most efficient. Another difficulty is the parasitic ionization of the background gas molecules that produces large amounts of undesirable ions of both polarities, preferentially positive. These ions are detected both directly and indirectly via ion-molecule reactions, which in both cases leads to abundant background and parasitic peaks, and thus limits the sensitivity. For helium that is most often used as a buffer gas, parasitic ionization occurs at electron energies exceeding 24 eV. Because of the low efficiency and high background, ion-electron reactions are not implemented on most analytical mass spectrometers.
For these reasons, it would be desirable to improve the efficiency of ion-electron reactions in mass spectrometric devices that utilize rf electric field.
The present invention provides devices and methods for producing effective ion-electron fragmentation reactions of positive and negative ions in multipolar radiofrequency fields used for storage and transportation of ions. An electron cloud is provided in the center of the field with kinetic electron energies below 20 eV, confined in radial direction by a magnetic field along the axis of the device.
In three-dimensional Paul ion traps with ring and end cap electrodes, the electrons are confined in radial direction by the magnetic field, and in axial direction by the electrical potential during a half period of the radiofrequency voltage; and means are provided for trapping the electrons in the direction along the axis of the device when the value of the radiofrequency voltage is positive. The electron cloud in the center can be provided at least once during every period of the radiofrequency, thus the duty cycle for ion-electron reactions can be 50% or higher.
In two-dimensional multipole field devices, like linear traps or ion guides, the magnetic confinement of the electrons in radial direction does not need to be supported by a confinement of the electrons in axial direction. The low kinetic energy electrons may freely drift along the axis of the device, or may be confined by a suitable force field like, e.g., a magnetic bottle.
Since the axial magnetic field prevents radial acceleration of the electrons by the radiofrequency voltage in both types of radiofrequency devices, the electrons essentially retain their initial kinetic energy during a significant part of the trapping period, and interact efficiently with the ions.